Reducing adapter dimer formation

ABSTRACT

Provided herein is a method of reducing adapter dimer formation comprising contacting a sample comprising target nucleic acid sequences with 5′ and 3′ adapters in the presence of one or more hairpin oligonucleotides. Also provided is a method of preparing a library of nucleic acid sequences comprising contacting first adapter oligonucleotides with a sample comprising target nucleic acid sequences under conditions to form first ligation products, contacting the sample with one or more hairpin oligonucleotides that binds to the first adapter oligonucleotides, and contacting the sample with second adapter oligonucleotides under conditions to bind to the first ligation products and form second ligation products, wherein the second ligation products form the library of nucleic acid sequences.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.61/409,867, filed Nov. 3, 2010, which is incorporated by referenceherein in its entirety.

BACKGROUND

Methods of creating libraries of nucleic acid molecules foramplification and/or sequencing techniques have been developed. Suchmethods include adding sequences (e.g., adapters) to the ends of targetnucleic acid sequences to facilitate amplification and/or sequencing ofthe target nucleic acid sequences. For example, adapters that containprimer sequences can be ligated onto the ends of target nucleic acidsequences. A single adapter or two different adapters can be used in theligation reaction. Such methods are known and described in, for example,WO 98/44151 and WO 00/18957, which are incorporated by reference hereinin their entireties. Such methods enable multiple target nucleic acidmolecules of the same or different, known or unknown sequence to beamplified in a single amplification reaction. Such target molecules canthen be used in, for example, sequencing techniques. However, a drawbackin preparing such libraries includes the formation of adapter-dimers.

SUMMARY

Provided herein is a method of reducing adapter dimer formationcomprising contacting a sample comprising target nucleic acid sequenceswith 5′ and 3′ adapters in the presence of one or more hairpinoligonucleotides under conditions to form 5′-adapter-target-3′-adaptersequences, wherein the amount of adapter dimer formation is reducedcompared to the amount of adapter dimer formation in the absence of thehairpin oligonucleotides. Also provided is a method of preparing alibrary of nucleic acid sequences comprising contacting first adapteroligonucleotides with a sample comprising target nucleic acid sequencesunder conditions to form first ligation products, contacting the samplewith one or more hairpin oligonucleotides that binds to the firstadapter oligonucleotides, and contacting the sample with second adapteroligonucleotides under conditions to bind to the first ligation productsand form second ligation products, wherein the second ligation productsform the library of nucleic acid sequences.

The details of one or more embodiments are set forth in the accompanyingdrawings and the description below. Other features, objects, andadvantages will be apparent from the description and drawings, and fromthe claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic showing adapter dimer formation during ligation of5′- and 3′-adapters to target nucleic acid sequences. Target nucleicacid sequences are ligated to a 3′-adapter followed by ligation to a5′-adapter. As shown in FIG. 1, addition of the 5′-adapter results inadapter dimers and 5′-adapter-target-3′-adapter sequences.

FIG. 2 is a schematic showing reduction of adapter dimer formation usinga hairpin oligonucleotide during ligation of 5′- and 3′-adapters totarget nucleic acid sequences.

FIG. 3 shows an exemplary hairpin oligonucleotide (SEQ ID NO:5) used inthe provided methods.

DETAILED DESCRIPTION

A major drawback in preparing libraries of target nucleic acids foramplification and/or sequencing by ligating adapters to the ends of thetarget nucleic acids is the formation of adapter dimers. Adapter dimersare formed by the ligation of two adapters directly to each other suchthat they do not contain a target nucleic acid insert (i.e., they do notform adapter-target-adapter nucleic acids). See, e.g., FIG. 1. Adapterdimers can reduce the efficiency of amplification reactions since theyare generally smaller than the adapter-target-adapter nucleic acids andaccumulate at a faster rate. Thus, amplification of theadapter-target-adapter nucleic acids is limited because the componentsof the amplification reaction, e.g., dNTPs and/or primers, are consumedby the adapter dimers. Similarly, in sequencing techniques, thesequencing of such adapter dimers reduces the efficiency of thetechnique since the adapter dimers fail to produce useful information asthey do not contain target nucleic acids. Thus, preparation of librariesof target nucleic acids with low levels of adapter dimers are desirable,e.g., for amplification and sequencing techniques, including when suchprocesses are high-throughput.

The present application provides methods of reducing adapter dimerformation. For example, the methods relate to preparation of librariesof target nucleic acid sequences in reduced amounts or in the absence ofadapter dimers. The present application also provides methods of usingsuch target nucleic acid sequences for amplification and/or sequencing.

The method of reducing adapter dimer formation includes contacting asample comprising target nucleic acid sequences with 5′ and 3′ adaptersin the presence of one or more hairpin oligonucleotides under conditionsto form 5′-adapter-target-3′-adapter sequences, wherein the amount ofadapter dimer formation is reduced compared to the amount in the absenceof the hairpin oligonucleotides. For example, less than 50, 45, 40, 35,30, 25, 20, 15, or 10% of the adapters form dimers in the providedmethods. Alternatively, at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,or 99% of the adapters do not form dimers in the provided methods.

Also provided is a method of preparing a library of nucleic acidsequences. The method includes contacting first adapter oligonucleotideswith a sample comprising target nucleic acid sequences under conditionsto form first ligation products, contacting the sample with one or morehairpin oligonucleotides that binds to the first adapteroligonucleotides, and contacting the sample with second adapteroligonucleotides under conditions to bind to the first ligation productsand form second ligation products, wherein the second ligation productsform the library of nucleic acid sequences. In particular embodiments,the 5′-end of the first adapters can be ligated to the 3′-end of thetarget nucleic acid sequences to form the first ligation products.Optionally, the 3′-end of the second adapters can be ligated to the5′-ends of the first ligation products to form the second ligationproducts. Optionally, the target nucleic acid molecules may comprisedifferent sequences.

In the provided methods, ligation of the first adapter to form the firstligation products can occur in the presence of a ligase and in theabsence of ATP. This is possible with the use of a pre-adenylatedadapter, which is described in more detail below.

The provided methods, optionally, further comprise isolating the secondligation products. Isolation of nucleic acids sequences is known andincludes, for example, gel electrophoresis. Suitable, standard methodsare also described in, for example, Sambrook and Russell, MolecularCloning: A Laboratory Manual, Third Edition (2001), which isincorporated by reference herein in its entirety. In particularembodiments, the provided methods further include amplifying and/orsequencing the second ligation products.

As used throughout, oligonucleotides or polynucleotide molecules includedeoxyribonucleic acids (DNA), ribonucleic acids (RNA) or other form ofnucleic acid. The polynucleotide molecule can be any form of natural,synthetic or modified DNA, including, but not limited to, genomic DNA,copy DNA, complementary DNA, or recombinant DNA. Alternatively, thepolynucleotide molecule can be any form of natural, synthetic ormodified RNA, including, but not limited to mRNA, ribosomal RNA,microRNA, siRNA or small nucleolar RNA. The polynucleotide molecule canbe partially or completely in double-stranded or single-stranded form.The terms “nucleic acid,” “nucleic acid molecule,” “oligonucleotide,”and “polynucleotide” are used interchangeably throughout. The differentterms are not intended to denote any particular difference in size,sequence, or other property unless specifically indicated otherwise. Forclarity of description the terms may be used to distinguish one speciesof molecule from another when describing a particular method orcomposition that includes several molecular species.

As used throughout, the term “target nucleic acid” refers to a nucleicacid molecule to which adapter oligonucleotides are ligated. Targetnucleic acid molecules can be any molecule to be amplified or sequenced.Target nucleic acids for use in the provided methods may be obtainedfrom any biological sample using known, routine methods. Suitablebiological samples include, but are not limited to, a blood sample,biopsy specimen, tissue explant, organ culture, biological fluid or anyother tissue or cell preparation, or fraction or derivative thereof orisolated therefrom. The biological sample can be a primary cell cultureor culture adapted cell line including but not limited to geneticallyengineered cell lines that may contain chromosomally integrated orepisomal recombinant nucleic acid sequences, immortalized orimmortalizable cell lines, somatic cell hybrid cell lines,differentiated or differentiatable cell lines, transformed cell lines,stem cells, germ cells (e.g. sperm, oocytes), transformed cell lines andthe like. For example, polynucleotide molecules may be obtained fromprimary cells, cell lines, freshly isolated cells or tissues, frozencells or tissues, paraffin embedded cells or tissues, fixed cells ortissues, and/or laser dissected cells or tissues. Biological samples canbe obtained from any subject or biological source including, forexample, human or non-human animals, including mammals and non-mammals,vertebrates and invertebrates, and may also be any multicellularorganism or single-celled organism such as a eukaryotic (includingplants and algae) or prokaryotic organism, archaeon, microorganisms(e.g. bacteria, archaea, fungi, protists, viruses), and aquaticplankton.

The target nucleic acid described herein can be of any length suitablefor use in the provided methods. For example, the target nucleic acidscan be at least 10, at least 20, at least 30, at least 40, at least 50,at least 75, at least 100, at least 150, at least 200, at least 250, atleast 500, or at least 1000 nucleotides in length or longer. Generally,if the target nucleic acid is a small RNA molecule, the target nucleicacid will be at least 10 nucleotides in length. Thus, the target nucleicacid sequences can comprise RNA molecules, for example, small RNAmolecules including, but not limited to miRNA molecules, siRNAmolecules, tRNA molecules, rRNA molecules, and combinations thereof. Insome embodiments, the target nucleic acid sequence comprisessingle-stranded DNA.

The provided target nucleic acids may be prepared to include 5′- and3′-adapters using a variety of standard techniques available and known.The adapters can be linear and can be double- or single-stranded.Exemplary methods of polynucleotide molecule preparation include, butare not limited to, those described in Bentley et al., Nature 456:49-51(2008); WO 2008/023179; U.S. Pat. No. 7,115,400; and U.S. PatentApplication Publication Nos. 2007/0128624; 2009/0226975; 2005/0100900;2005/0059048; 2007/0110638; and 2007/0128624, each of which is hereinincorporated by reference in its entirety. Target nucleic acids aremodified to comprise one or more regions of known sequence (e.g., anadapter) located on the 5′ and 3′ ends. Optionally, the adaptercomprises the indexing tag. When the target nucleic acid moleculescomprise known sequences on the 5′ and 3′ ends, the known sequences canbe the same or different sequences. Optionally, as described more fullybelow, a known sequence located on the 5′ and/or 3′ ends of thepolynucleotide molecules is capable of hybridizing to one or moreoligonucleotides immobilized on a surface. For example, a polynucleotidemolecule comprising a 5′ known sequence may hybridize to a firstplurality of oligonucleotides while the 3′ known sequence may hybridizeto a second plurality of oligonucleotides.

The adapters that are added to the 5′ and/or 3′ end of a nucleic acidcan comprise a universal sequence. A universal sequence is a region ofnucleotide sequence that is common to, i.e., shared by, two or morenucleic acid molecules. Optionally, the two or more nucleic acidmolecules also have regions of sequence differences. Thus, for example,the 5′ adapters can comprise identical or universal nucleic acidsequences and the 3′ adapters can comprise identical or universalsequences. A universal sequence that may be present in different membersof a plurality of nucleic acid molecules can allow the replication oramplification of multiple different sequences using a single universalprimer that is complementary to the universal sequence. Similarly, atleast one, two (e.g., a pair) or more universal sequences that may bepresent in different members of a collection of nucleic acid moleculescan allow the replication or amplification of multiple differentsequences using at least one, two (e.g., a pair) or more singleuniversal primers that are complementary to the universal sequences.Thus, a universal primer includes a sequence that can hybridizespecifically to such a universal sequence. The target nucleic acidsequence-bearing molecules may be modified to attach universal adapters(e.g., non-target nucleic acid sequences) to one or both ends of thedifferent target nucleic acid sequences, the adapters providing sitesfor hybridization of universal primers. This approach has the advantagethat it is not necessary to design a specific pair of primers for eachtemplate to be generated, amplified, sequenced, and/or otherwiseanalyzed; a single pair of primers can be used for amplification ofdifferent templates provided that each template is modified by additionof the same universal primer-binding sequences to its 5′ and 3′ ends.

In particular embodiments, the 3′-adapter or first adapteroligonucleotide is a 3′-pre-adenylated adapter. Pre-adenylated adaptersare known and are described in, for example, U.S. Publication No.2010/0062494 and U.S. Publication No. 2009/0011422, which areincorporated by reference herein in their entireties. Exemplary3′-pre-adenylated adapter sequences include, but are not limited to,rAppTGGAATTCTCGGGTGCCAAGG (SEQ ID NO:13) Optionally, the 5′-adapter orsecond adapter oligonucleotide is a 5′-RNA adapter. Optionally, the5′-RNA adapter is GUUCAGAGUUCUACAGUCCGACGAUC (SEQ ID NO:14).

As used throughout, the phrases adapter-target-adapter library orlibrary of adapter-target-adapters refers to a collection or pluralityof adapter-target-adapter molecules that share common or universalsequences at their 5′ ends and common or universal sequences at their 3′ends. The sequences at the 5′ and 3′ ends of the adapter-target-adapterscan be the same or different. Similarly, the target nucleic acidsequences in the adapter-target-adapters can be the same or different.

As used throughout, the term “hairpin oligonucleotide” refers to anucleic acid sequence that has two complementary regions that hybridizeto one another to form a double-stranded region with the twocomplementary regions being connected by a single-stranded loop. Hairpinoligonucleotides used in the provided methods have additionalnucleotides at the 5′ end forming a 5′ overhanging sequence portion. Inparticular embodiments, the 5′ overhanging sequence portion binds to oneof the adapters to reduce or eliminate adapter dimer formation. Forexample, the single-stranded portion of the hairpin oligonucleotide cananneal to the adapter (e.g., the first or 3′-adapter). Thus, inparticular embodiments, the hairpin oligonucleotide is designed suchthat the 5′ overhang sequence is complementary to, at least, a portionof an adapter sequence so that the hairpin can anneal to the adapter.Optionally, the hairpin oligonucleotide is not phosphorylated at its5′-end such that the hairpin oligonucleotide will not ligate to thesecond or 5′-adapter. In particular embodiments, the hairpinoligonucleotides are, optionally, not complementary to the targetnucleic acid sequences. Exemplary hairpin oligonucleotides that can beused in the provided methods include, but are not limited to SEQ ID NOs:2-12 and 15-25.

Suitable concentrations of hairpin oligonucleotides for use in theprovided methods can be readily determined by those of skill in the art.By way of example, the ratio of hairpin to adapter can be 10:1, 9:1,8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 and 1:1. Optionally, the ratio ofhairpin to adapter is between 4:1 and 1:1.

The hairpin oligonucleotides described herein can be of any lengthsuitable for use in the provided methods. For example, the hairpinoligonucleoties can be at least 10, at least 20, at least 30, at least40, or at least 50, nucleotides in length or longer. Optionally, thehairpin oligonucleotides are 15 to 40 base pairs in length.

Optionally, the hairpin and 3′- or first adapter complexes are removedprior to ligation of the 5′- or second adapters to the target-3′-adapteror first ligation products. This can be carried out, for example, byaffinity capture. In affinity capture, one member of a binding pair maybe attached to hairpin oligonucleotide and the other member of thebinding pair can be attached to a surface, e.g., a membrane, column orbead. Thus, the hairpin oligonucleotides may be labeled directly orindirectly. By way of example, the hairpins may be biotinylated (e.g.,using enzymatic incorporation of biotinylated nucleotides). Biotinylatedhairpin molecules can then be captured on streptavidin-coated beads.Similarly, other hapten-receptor combinations can be used, including,but not limited to, digoxigenin and anti-digoxigenin antibodies or2,4-dinitrophenol (DNP) and anti-DNP. Fluorogens can also be used tomodify the hairpin oligonucleotides. Examples of fluorogens includefluorescein and derivatives, phycoerythrin, allo-phycocyanin,phycocyanin, rhodamine, TEXAS RED® (Molecular Probes, Inc., Eugene,Oreg.) or other fluorogens. The fluorogens are generally attached bychemical modification and bind to a fluorogen-specific antibody, such asanti-fluorescein. It will be understood nucleic acids can also be taggedby incorporation of a modified base containing any chemical grouprecognizable by specific antibodies. Other tags and methods of taggingnucleotide sequences for capture onto a surface coated with substrateare well known. For example, a review of nucleic acid labels can befound in the article by Landegren, et al. “DNA Diagnostics-MolecularTechniques and Automation”, Science, 242:229-237 (1988), which isincorporated herein by reference in its entirety.

As described above, the hairpin oligonucleotides can be labeled and canbind to a surface, including, but not limited to, a column or bead, suchas, for example, a magnetic bead. If the label is biotin, the beads(e.g., magnetic beads) are streptavidin-coated beads. As used herein, asurface includes any solid surface or matrix to which the labeledhairpin oligonucleotide can bind. Suitable solid surfaces or matricesinclude, for example, beads, resins, gels, wells, columns, chips,flowcells, membranes, matrices, plates or filters. Solid surfaces can beany solid plastic material in the shape of plates, slides, dishes,beads, particles, cups, strands, chips and strips. Solid surfaces alsoinclude glass beads, glass test tubes and any other appropriate glassproduct.

The target nucleic acid molecules can be modified to include any nucleicacid sequence desirable using standard, known methods. Such additionalsequences may include, for example, restriction enzyme sites, oroligonucleotide indexing tag in order to permit identification ofamplification products of a given nucleic acid sequence. As describedherein, the indexing tag can be added to a polynucleotide molecule byinclusion on an adapter or on a primer. Optionally, the indexing tag canbe directly ligated to the ends of a polynucleotide molecule.

Once a library of nucleic acids (e.g., a library ofadapter-target-adapters) has been generated, the nucleic acids can beamplified and/or sequenced. Nucleic acid amplification includes theprocess of amplifying or increasing the numbers of a nucleic acidtemplate and/or of a complement thereof that are present, by producingone or more copies of the template and/or or its complement.Amplification can be carried out by a variety of known methods underconditions including, but not limited to, thermocycling amplification orisotheraml amplification. For example, methods for carrying outamplification are described in U.S. Publication No. 2009/0226975; WO98/44151; WO 00/18957; WO 02/46456; WO 06/064199; and WO 07/010,251;which are incorporated by reference herein in their entireties. Thus,amplification can occur on the surface to which the nucleic acidmolecules are attached. This type of amplification can be referred to assolid phase amplification, which when used in reference to nucleicacids, refers to any nucleic acid amplification reaction carried out onor in association with a surface (e.g., a solid support). For example,all or a portion of the amplified products are synthesized by extensionof an immobilized primer. Solid phase amplification reactions areanalogous to standard solution phase amplifications except that at leastone of the amplification oligonucleotides is immobilized on a surface(e.g., a solid support).

Solid-phase amplification may comprise a nucleic acid amplificationreaction comprising only one species of oligonucleotide primerimmobilized to a surface. Alternatively, the surface may comprise aplurality of first and second different immobilized oligonucleotideprimer species. Solid-phase amplification may comprise a nucleic acidamplification reaction comprising one species of oligonucleotide primerimmobilized on a solid surface and a second different oligonucleotideprimer species in solution. Solid phase nucleic acid amplificationreactions generally comprise at least one of two different types ofnucleic acid amplification, interfacial and surface (or bridge)amplification. For instance, in interfacial amplification, the solidsupport comprises a template nucleic acid molecule that is indirectlyimmobilized to the solid support by hybridization to an immobilizedoligonucleotide primer, the immobilized primer may be extended in thecourse of a polymerase-catalyzed, template-directed elongation reaction(e.g., primer extension) to generate an immobilized polynucleotidemolecule that remains attached to the solid support. After the extensionphase, the nucleic acids (e.g., template and its complementary product)are denatured such that the template nucleic acid molecule is releasedinto solution and made available for hybridization to anotherimmobilized oligonucleotide primer. The template nucleic acid moleculemay be made available in 1, 2, 3, 4, 5 or more rounds of primerextension or may be washed out of the reaction after 1, 2, 3, 4, 5 ormore rounds of primer extension.

In surface (or bridge) amplification, an immobilized nucleic acidmolecule hybridizes to an immobilized oligonucleotide primer. The 3′ endof the immobilized nucleic acid molecule provides the template for apolymerase-catalyzed, template-directed elongation reaction (e.g.,primer extension) extending from the immobilized oligonucleotide primer.The resulting double-stranded product “bridges” the two primers and bothstrands are covalently attached to the support. In the next cycle,following denaturation that yields a pair of single strands (theimmobilized template and the extended-primer product) immobilized to thesolid support, both immobilized strands can serve as templates for newprimer extension.

Optionally, amplification of the adapter-target-adapters or library ofnucleic acid sequences results in clustered arrays of nucleic acidcolonies, analogous to those described in U.S. Pat. No. 7,115,400; U.S.Publication No. 2005/0100900; WO 00/18957; and WO 98/44151, which areincorporated by reference herein in their entireties. Clusters andcolonies are used interchangeably and refer to a plurality of copies ofa nucleic acid sequence and/or complements thereof attached to asurface. Typically, the cluster comprises a plurality of copies of anucleic acid sequence and/or complements thereof, attached via their 5′termini to the surface. The copies of nucleic acid sequences making upthe clusters may be in a single or double stranded form.

Clusters may be detected, for example, using a suitable imaging means,such as, a confocal imaging device or a charge coupled device (CCD)camera. Exemplary imaging devices include, but are not limited to, thosedescribed in U.S. Pat. Nos. 7,329,860; 5,754,291; and 5,981,956; and WO2007/123744, each of which is herein incorporated by reference in itsentirety. The imaging means may be used to determine a referenceposition in a cluster or in a plurality of clusters on the surface, suchas the location, boundary, diameter, area, shape, overlap and/or centerof one or a plurality of clusters (and/or of a detectable signaloriginating therefrom). Such a reference position may be recorded,documented, annotated, converted into an interpretable signal, or thelike, to yield meaningful information. The signal may, for instance,take the form of a detectable optical signal emanating from a definedand identifiable location, such as a fluorescent signal, or may be adetectable signal originating from any other detectable label asprovided herein. The reference position of a signal generated from twoor more clusters may be used to determine the actual physical positionon the surface of two clusters that are related by way of being thesites for simultaneous sequence reads from different portions of acommon target nucleic acid.

Following amplification, the adapter-target-adapters or library ofnucleic acids can be sequenced. The sequencing is carried out by avariety of known methods, including, but not limited to, sequencing byligation, sequencing by synthesis or sequencing by hybridization.

Sequencing by synthesis, for example, is a technique wherein nucleotidesare added successively to a free 3′ hydroxyl group, typically providedby annealing of an oligonucleotide primer (e.g., a sequencing primer),resulting in synthesis of a nucleic acid chain in the 5′ to 3′direction. These and other sequencing reactions may be conducted on theherein described surfaces bearing nucleic acid clusters. The reactionscomprise one or a plurality of sequencing steps, each step comprisingdetermining the nucleotide incorporated into a nucleic acid chain andidentifying the position of the incorporated nucleotide on the surface.The nucleotides incorporated into the nucleic acid chain may bedescribed as sequencing nucleotides and may comprise one or moredetectable labels. Suitable detectable labels, include, but are notlimited to, haptens, radionucleotides, enzymes, fluorescent labels,chemiluminescent labels, and/or chromogenic agents. One method fordetecting fluorescently labeled nucleotides comprises using laser lightof a wavelength specific for the labeled nucleotides, or the use ofother suitable sources of illumination. The fluorescence from the labelon the nucleotide may be detected by a CCD camera or other suitabledetection means. Suitable instrumentation for recording images ofclustered arrays is described in WO 07/123,744, the contents of whichare incorporated herein by reference herein in its entirety.

Optionally, cycle sequencing is accomplished by stepwise addition ofreversible terminator nucleotides containing, for example, a cleavableor photobleachable dye label as described, for example, in U.S. Pat.Nos. 7,427,673; 7,414,116; WO 04/018497; WO 91/06678; WO 07/123,744; andU.S. Pat. No. 7,057,026, the disclosures of which are incorporatedherein by reference in their entireties. The availability offluorescently-labeled terminators in which both the termination can bereversed and the fluorescent label cleaved facilitates efficient cyclicreversible termination (CRT) sequencing. Polymerases can also beco-engineered to efficiently incorporate and extend from these modifiednucleotides.

Alternatively, pyrosequencing techniques may be employed. Pyrosequencingdetects the release of inorganic pyrophosphate (PPi) as particularnucleotides are incorporated into the nascent strand (Ronaghi et al.,(1996) “Real-time DNA sequencing using detection of pyrophosphaterelease.” Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001)“Pyrosequencing sheds light on DNA sequencing.” Genome Res. 11(1), 3-11;Ronaghi, M., Uhlen, M. and Nyren, P. (1998) “A sequencing method basedon real-time pyrophosphate.” Science 281(5375), 363; U.S. Pat. Nos.6,210,891; 6,258,568; and 6,274,320, the disclosures of which areincorporated herein by reference in their entireties). Inpyrosequencing, released PPi can be detected by being immediatelyconverted to adenosine triphosphate (ATP) by ATP sulfurylase, and thelevel of ATP generated is detected via luciferase-produced photons.

Additional exemplary sequencing-by-synthesis methods that can be usedwith the methods described herein include those described in U.S. PatentPublication Nos. 2007/0166705; 2006/0188901; 2006/0240439; 2006/0281109;2005/0100900; U.S. Pat. No. 7,057,026; WO 05/065814; WO 06/064199; WO07/010,251, the disclosures of which are incorporated herein byreference in their entireties.

Alternatively, sequencing by ligation techniques are used. Suchtechniques use DNA ligase to incorporate oligonucleotides and identifythe incorporation of such oligonucleotides and are described in U.S.Pat. Nos. 6,969,488; 6,172,218; and 6,306,597; the disclosures of whichare incorporated herein by reference in their entireties. Other suitablealternative techniques include, for example, fluorescent in situsequencing (FISSEQ), and Massively Parallel Signature Sequencing (MPSS).

Also provided are kits for reducing adapter dimer formation includinginclude one or more of the provided hairpin oligonucleotides. The kitscan also include 5′- and/or 3′-adapters. Optionally, the kits alsoinclude suitable primers of appropriate nucleotide sequence for use withthe adapters. The kits may also include buffers, enzymes, such as, forexample, a ligase or polymerase, dNTPs, and the like.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation may not be explicitly disclosed,each is specifically contemplated and described herein. For example, ifa method is disclosed and discussed and a number of modifications thatcan be made to the method steps are discussed, each and everycombination and permutation of the method steps, and the modificationsthat are possible are specifically contemplated unless specificallyindicated to the contrary. Likewise, any subset or combination of theseis also specifically contemplated and disclosed. This concept applies toall aspects of this disclosure. Thus, if there are a variety ofadditional steps that can be performed it is understood that each ofthese additional steps can be performed with any specific method stepsor combination of method steps of the disclosed methods, and that eachsuch combination or subset of combinations is specifically contemplatedand should be considered disclosed.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the claims.

EXAMPLE Example 1 Reducing Adapter Dimer Formation Using HairpinOligonucleotides

Using total RNA as input, 1 μg of total RNA was added to the firstligation reaction tube containing water and the 3′-pre-adenylatedadapter. 5× HM ligation buffer, RNase inhibitor and truncated T4 RNAligase was then added to the reaction tube and the tube incubated at 28°C. for one hour. The blocker was then added to the reaction tube andincubated at 28° C. for fifteen minutes and then the tube was placed onice. The 5′-adapter, 10 mM ATP, and T4 RNA ligase was then added to thereaction tube and incubated at 28° C. for one hour. Reversetranscription primer, DTT, 5× First Stand Buffer, 12.5 mM dNTPs, RNaseInhibitor and Superscript II Reverse Transcriptase was added to thereaction tube and incubated at 50° C. for an hour. Ultrapure water, 5×Phusion HF Buffer, 2 PCR primers, Phusion DNA polymerase and 25 mM dNTPswas added to the reaction and the reaction was amplified under thefollowing conditions: 30 seconds at 98° C., 40 cycles of 10 seconds at98° C., 30 seconds at 60° C., 15 seconds at 72° C., and 10 minutes at72° C. Following PCR, the products were analyzed for percent adapterdimer formation. The results are shown in Table 1. A variety of blockerswere used, an RNA linear blocker (SEQ ID NO:1), four different RNAhairpin blockers (SEQ ID NOs:2-5), and 7 different chimera blockerscomprising RNA and DNA sequences (SEQ ID NOs:6-12).

TABLE 1 Percent Adapter Dimer Formation Percent Blocker Concentration(μM) Adapter Dimer* None 0.0 78.9 Linear Blocker 1.6 81.4 (SEQ ID NO: 1)Blocker 1 (SEQ ID NO: 2) 3.2 41.3 Blocker 1 6.4 41.8 Blocker 2 (SEQ IDNO: 3) 3.2 41.5 Blocker 2 6.4 39.9 Blocker 3 (SEQ ID NO: 4) 3.2 27.6Blocker 3 6.4 17.2 Blocker 4 (SEQ ID NO: 5) 0.8 16.8 Blocker 4 1.6 17.7Blocker 4 3.2 18.8 Blocker 4 6.4 19.9 Blocker 4 12.8 23.7 ChimeraBlocker 1 3.2 87.2 (SEQ ID NO: 6) Chimera Blocker 2 3.2 90.1 (SEQ ID NO:7) Chimera Blocker 3 3.2 22.4 (SEQ ID NO: 8) Chimera Blocker 4 3.2 16.9(SEQ ID NO: 9) Chimera Blocker 5 3.2 58.0 (SEQ ID NO: 10) ChimeraBlocker 6 3.2 13.3 (SEQ ID NO: 11) Chimera Blocker 7 3.2 58.3 (SEQ IDNO: 12) *Average of at least two experiments

SEQUENCES: SEQ ID NO: 1 - LINEAR BLOCKER (RNA)UACGUGCAAGAGUCCUACAGACGGUCAUCCGGGCCUCACCAUACAAUCACSEQ ID NO: 2 - HAIRPIN BLOCKER 1 (RNA) UUCCAACGAUUCAACGUSEQ ID NO: 3 - HAIRPIN BLOCKER 2 (RNA) UUCCAACGUUCCCGUSEQ ID NO: 4 - HAIRPIN BLOCKER 3 (RNA) UUCCACCACGAUUCAACGUGGSEQ ID NO: 5 - HAIRPIN BLOCKER 4 (RNA) GAAUUCCACCACGUUCCCGUGGSEQ ID NO: 6 - HAIRPIN CHIMERA BLOCKER 1 (RNA/DNA)GAATTCCACCACGTTCCrCrGrUrGrG SEQ ID NO: 7 - HAIRPIN CHIMERA BLOCKER 2(RNA/DNA) TTCCACCACGATTCAArCrGrUrGrGSEQ ID NO: 8 - HAIRPIN CHIMERA BLOCKER 3 (RNA/DNA) TTCCAACGTTCCrCrGrUSEQ ID NO: 9 - HAIRPIN CHIMERA BLOCKER 4 (RNA/DNA) TTCCAACGATTCAArCrGrUSEQ ID NO: 10 - HAIRPIN CHIMERA BLOCKER 5 (RNA/DNA)TTCCACCACGATTCAACrGrUrGrG SEQ ID NO: 11 - HAIRPIN CHIMERA BLOCKER 6(RNA/DNA) GAATTCCACCACGTTCCCGrUrGrGSEQ ID NO: 12 - HAIRPIN CHIMERA BLOCKER 7 (RNA/DNA)TTCCACCACGATTCAACGrUrGrG SEQ ID NO: 15 - HAIRPIN BLOCKER 5 (RNA)GAAUUCCACCACGUUUUCCCGUGG SEQ ID NO: 16 - HAIRPIN BLOCKER 6 (RNA)GAAUUCCACCACGUCUCCCCGUGG SEQ ID NO: 17 - HAIRPIN BLOCKER 7 (RNA)GAAUUCCACCACGUCUCUCCGUGG SEQ ID NO: 18 - HAIRPIN BLOCKER 8 (RNA)GAAUUCCACCACGUGUGUGCGUGG SEQ ID NO: 19 - HAIRPIN BLOCKER 9 (RNA)GAAUUCCACCACGUGUGUGCGUGG SEQ ID NO: 20 - HAIRPIN BLOCKER 10 (RNA)GAAUUCCACCACGUUUUCGUGG SEQ ID NO: 21 - HAIRPIN BLOCKER 11 (RNA)GAGAAUUCCACCACGUUCCCGUGG SEQ ID NO: 22 - HAIRPIN BLOCKER 12 (RNA)CCGAGAAUUCCACCACGUUCCCGUGG SEQ ID NO: 23 - HAIRPIN BLOCKER 13 (RNA)GAAUUCCACCACGCUUCCGCGUGG SEQ ID NO: 24 - HAIRPIN BLOCKER 14 (RNA)GAAUUCCACCACGCGUUCCCGCGUGG SEQ ID NO: 25 - HAIRPIN BLOCKER 15 (RNA)ACCCGAGAAUUCCACCACGUUCCCGUGG

What is claimed is:
 1. A method of preparing a library of nucleic acidsequences comprising: (a) contacting first adapter oligonucleotides witha sample comprising target nucleic acid sequences under conditions toform first ligation products; (b) after step (a), contacting the samplewith one or more hairpin oligonucleotides having a 5′ overhang portionconfigured to bind unligated first adaptor oligonucleotides whereby said5′ overhang portion is complementary to and anneals to at least aportion of said unligated first adapter oligonucleotides; wherein thehairpin oligonucleotides do not anneal to the first ligation products;(c) after step (b), contacting the sample with second adapteroligonucleotides under conditions to bind to the first ligation productsand form second ligation products, wherein the second ligation productsform the library of nucleic acid sequences; and wherein the first andsecond adapters are linear and single-stranded.
 2. The method of claim1, wherein step (a) occurs in the presence of a ligase and in theabsence of ATP.
 3. The method of claim 1, further comprising isolatingthe second ligation products.
 4. The method of claim 3, wherein theisolation comprises gel electrophoresis.
 5. The method of claim 1,further comprising amplifying the second ligation products.
 6. Themethod of claim 1, further comprising sequencing the second ligationproducts.
 7. The method of claim 1, wherein the target nucleic acidsequences comprise RNA molecules.
 8. The method of claim 7, wherein theRNA molecules comprise small RNA molecules.
 9. The method of claim 8,wherein the small RNA molecules comprise molecules selected from thegroup consisting of miRNA molecules, siRNA molecules, tRNA molecules,rRNA molecules, and combinations thereof.
 10. The method of claim 1,wherein the target nucleic acid sequence comprises single-stranded DNA.11. The method of claim 1, wherein the first adapter is a3′-pre-adenylated adapter.
 12. The method of claim 1, wherein the secondadapter is a 5′-RNA adapter.
 13. The method of claim 1, wherein thehairpin oligonucleotide comprises RNA.
 14. The method of claim 1,wherein the hairpin oligonucleotide comprises a label.
 15. The method ofclaim 14, wherein the label binds a column or bead.
 16. The method ofclaim 15, wherein the bead is a magnetic bead.